This application claims priority to Japanese Application Serial No. 361237/2000, filed Nov. 28, 2000.
The present invention relates to a method and an apparatus for determining an amount of light for each of a plurality of microspots arranged on a plane. More particularly, the present invention relates to a method and an apparatus for reading fluorescence on a biochip where biological substances such as DNAs or proteins labeled with a time-resolved fluorescent substance are arranged as a high-density spot array.
Currently-practiced methods for analyzing chemical and physical properties of biological substances such as DNA and proteins often utilize fluorescence. These methods use a biochip on which biological substances such as DNAs or proteins that are labeled with a fluorescent substance marker are arranged as a high-density microspot array. In order to read these spots, a fluorescence reading apparatus is necessary which scans the spots with a laser beam to excite the fluorescent label existing in each spot on the biochip, thereby reading the excited fluorescence from each spot.
FIG. 7 is a schematic view of a conventional fluorescence reading apparatus. A biochip 101 is a rectangular glass plate whose surface is provided with DNAs labeled with fluorescent substance Cy3 (excitation wavelength: 552 nm, fluorescence wavelength: 565 nm, duration: 1.3 ns) which are aligned as microspots in a matrix along x- and y-directions. The biochip 101 is placed on a stage 121 which travels stepwisely in the y-direction by a y-direction driving motor 103. A reading head 111 placed above the biochip 101 is continuously driven in the x-direction by a x-direction driving motor 114. A laser beam 110 emitted from a laser light source 104 is radiated on the biochip 101 via the reading head 111. The fluorescence generated on the biochip 101 is captured by a photomultiplier 116 via the reading head 111. The laser light source 104 emits a laser beam at a wavelength of 552 nm, a wavelength appropriate to excite the marker Cy3.
Upon reading the biochip 101, the reading head 111 is continuously transferred in the x-direction along an x-axis rail 122 by the x-direction driving motor 114 under the control of a control computer 108. Similarly, the stage 121 holding the biochip 101 is transferred stepwisely in the y-direction along a y-axis rail 123 under the control of the control computer 108.
FIGS. 8A to 8E are schematic diagrams showing the order for reading the respective spots on the biochip 101 according to a conventional reading. The spots are arranged in a two-dimensional matrix along x- and y-directions. A black dot represents a spot whose fluorescence has been read, while a white dot represents a spot whose fluorescence has not yet been read.
As shown in FIGS. 8A to 8C, the spots 119 on the biochip 101 are irradiated with the laser beam at a constant rate. Irradiation by the laser beam as well as reading the fluorescence resulting from the laser beam irradiation take place simultaneously and continuously. As shown in FIG. 8C, once scanning for a single row in the x-direction is completed, the reading head 111 returns to the left end of the same row. Then, the stage 121 is transferred stepwisely to send the biochip 101 in the y-direction for one resolution distance. Then, as shown in FIGS. 8D and 8E, the next row is scanned continuously in the x-direction. By repeating this series of steps, the entire area of the biochip 101 is completely scanned.
When a fluorescent substance such as Cy3 is used to label a biological substance, fluorescence should be read while radiating excitation light since the duration of fluorescence is as short as a few ns. Although the fluorescence capturing member is provided with an optical filter or the like that only passes the wavelength of the fluorescence and blocks the excitation light, it is hard to detect only the fluorescence by completely blocking the excitation light since the fluorescent intensity of the fluorescent label is as extremely weak as about {fraction (1/1000000)}the intensity of the excitation laser beam.
Thus, a time-resolved fluorescent substance such as europium (duration: 400 xcexcs) with a very long fluorescent duration as compared to, for example, Cy3 (duration: 1.3 ns) may be used. The time-resolved fluorescent substance excited by laser beam irradiation retains the excitation state even after the laser beam irradiation depending on the relative duration. By utilizing this property, the laser beam irradiation and the fluorescence reading may be performed asynchronously so that fluorescence reading takes place after the excitation light irradiation, thereby preventing deterioration of the S/N ratio caused by capturing the excitation laser beam during the fluorescence reading. However, even when such a time-resolved fluorescent substance is employed as a label, the S/N ratio of the fluorescence detection of the spots can be poor since the conventional fluorescence reading apparatus sequentially reads the time-resolved fluorescent substances on the biochip along the laser beam irradiation path, and thus residual fluorescence resulting from the longer duration may become a noise and interfere with the reading of the adjacent spot.
In view of the above-mentioned problems, the present invention has an objective of providing a method and an apparatus for reading fluorescence, with which deterioration of the S/N ratio caused by residual fluorescence from an adjacent spot resulting from the use of the time-resolved fluorescent label can be prevented, thereby realizing fluorescence reading at a high S/N ratio.
In order to achieve the above-mentioned objective, the method for reading fluorescence of the present invention reads fluorescence by radiating excitation light on a plurality of spots arranged at predetermined intervals each containing a substance labeled with a time-resolved fluorescent substance and detecting fluorescence generated from each of the plurality of spots, the method comprising: a first step of irradiating a first spot with excitation light for a predetermined time; a second step of detecting fluorescence generated from the first spot after the irradiation with the excitation light; a third step of irradiating a second spot with excitation light, which is distanced from the first spot by a distance that is two or more times the distance between adjacent spots; and a fourth step of detecting fluorescence generated from the second spot after the irradiation with the excitation light, wherein, the third and fourth steps are repeated to detect fluorescence from all of the spots.
The plurality of spots may be arranged in a two-dimensional matrix along x- and y-directions, in a concentric pattern or in a spiral pattern. A second spot, which is distanced from the first spot by a distance two or more times the distance between the spots, is typically two spots away from the first spot that has just underwent fluorescence detection.
The time-resolved fluorescent substance according to the present invention refers to a fluorescent substance that lasts for a duration of 10 ns or longer. Examples of the time-resolved fluorescent substance include europium (excitation wavelength: 326 nm, fluorescence wavelength: 612 nm, duration: 400 xcexcs) and luciferin. In order to enhance the detection sensitivity, longer duration is desirable.
According to above-described method, undesirable capture of the excitation light as well as undesirable capture of the time-resolved fluorescence emitted from already-detected spots can be avoided, thereby greatly improving the SIN ratio of fluorescence detection.
A method according to the present invention reads fluorescence by radiating excitation light on a plurality of spots arranged in a line at predetermined intervals each containing a substance labeled with a time-resolved fluorescent substance and detecting fluorescence generated from each of the plurality of spots, wherein an operation of radiating excitation light to a spot for a predetermined time and thereafter detecting fluorescence generated from the spot is performed sequentially for every nth spots (where n is an integer of 2 or higher), and when the operation reaches the end of the line, the operation goes back towards the top of the line and is repeated for every nth spots (where n is an integer of 2 or higher) starting from a spot that has not yet undergone fluorescence detection.
Furthermore, a method according to the present invention reads fluorescence by radiating excitation light on a plurality of spots arranged in a line at predetermined intervals each containing a substance labeled with a time-resolved fluorescent substance and detecting fluorescence generated from each of the plurality of spots, wherein an operation of radiating excitation light to a spot for a predetermined time and thereafter detecting fluorescence generated from the spot is performed sequentially for every second spots, and when the operation reaches the end of the line, the operation goes back towards the top of the line and is repeated for the remaining spots.
According to the above-described method for reading fluorescence, the substance labeled with the time-resolved fluorescent substance may be a biological substance.
An apparatus of the present invention reads fluorescence by detecting fluorescence from a plurality of spots arranged on a substrate each containing a substance labeled with a time-resolved fluorescent substance, the apparatus comprising: a stage for holding the substrate; a laser light source; a reading head movable in a two-dimensional manner relative to the substrate, the reading head provided with an excitation light optical system for irradiating the spots on the substrate with a laser beam emitted from the laser light source and a light capturing optical system for capturing fluorescence generated from the spots; an optical detector for detecting fluorescence captured by the light capturing optical system; a driver for driving the stage and the reading head relative to each other; and a controller for controlling the laser light source, the light detector and the driver, wherein the controller controls the driver such that the reading head is aligned above each of the spots on the substrate in a skipping manner, and controls the laser light source and the light detector such that a spot below the reading head is irradiated with a laser beam for a predetermined time and thereafter fluorescence generated from the irradiated spot is detected.
Alignment in a skipping manner means to first target one spot to perform fluorescence detection, and then to target a next spot that is not directly adjacent to the first spot (typically, one after the spot directly adjacent to the first spot) to perform fluorescence detection. Owing to this skipping alignment (fluorescence detection in a skipping manner), capturing the excitation light as well as capturing the time-resolved fluorescence from already-detected spot can be avoided, thereby greatly enhancing the S/N ratio of the fluorescence detection.
Preferably, the irradiation light optical system and the light capturing optical system form a confocal optical system.
The driver may comprise a reading head driver for driving the reading head in one direction, and a stage driver for driving the stage in a direction generally perpendicular to the driving direction of the reading head.
The fluorescence reading apparatus of the invention is useful for reading a biochip provided with a plurality of spots containing biological substances labeled with a time-resolved fluorescent substance.